ANTI-PCSK9 AND METHODS FOR TREATING LIPID AND CHOLESTEROL DISORDERS

• US 20110033465A1
• Filed: 10/27/2008
• Published: 02/10/2011
• Est. Priority Date: 10/26/2007
• Status: Active Grant

• Current Assignee: Merck Sharp & Dohme LLC (Merck & Co., Inc.)
• Original Assignee: Schering Corporation (Merck & Co., Inc.)
• Inventors: Hedrick, Joseph A., Hollenbaugh, Diane, Monsma, Frederick James JR., Churakova, Tatyana
• Granted Patent: US 8,598,320 B2
• Time in Patent Office: Days
• US Class Current: 424/136.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 31/397 having four-membered rings, e.g. azetidine
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Abstract

The present invention provides compositions and methods for treating disorders of cholesterol and lipid metabolism by administration of an anti-PCSK9 antibody or a peptide inhibitor of PCSK9.

Claims

1. An isolated polypeptide comprising a member selected from the group consisting of

(i) CDR-H1, CDR-H2 and CDR-H3 of the variable region the 11B5 heavy chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO:

10;

(ii) CDR-H1, CDR-H2 and CDR-H3 of the variable region the 75B9 heavy chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO;

18;

(iii) CDR-H1, CDR-H2 and CDR-H3 of the variable region the 77D10 heavy chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO;

26;

(iv) CDR-H1, CDR-H2 and CDR-H3 of the variable region the 29C10 heavy chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO;

34;

(v) CDR-H1, CDR-H2 and CDR-H3 of the variable region the 22D11 heavy chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO;

42;

(vi) CDR-H1, CDR-H2 and CDR-H3 of the variable region the 1F11/1G11 heavy chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO;

50;

(vii) CDR-L1, CDR-L2 and CDR-L3 of the variable region the 11B5 light chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO;

14;

(viii) CDR-L1, CDR-L2 and CDR-L3 of the variable region the 7559 light chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO;

22;

(ix) CDR-L1, CDR-L2 and CDR-L3 of the variable region the 77D10 light chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO;

30;

(x) CDR-L1, CDR-L2 and CDR-L3 of the variable region the 29C10 light chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO;

38;

(xi) CDR-L1, CDR-L2 and CDR-L3 of the variable region the 22D11 light chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO;

46; and

(xii) CDR-L1, CDR-L2 and CDR-L3 of the variable region the 1F11/1G11 light chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO;

54.

2. The polypeptide of claim 1 comprising CDR-H1, CDR-H2 and CDR-H3 of the variable region in the 1F11/1G11 heavy chain immunoglobulin which variable region comprises the amino acid sequence of SEQ ID NO:

50.

6. An isolated antibody or antigen-binding fragment thereof comprising the polypeptide of claim 2.

7. The antibody or antigen-binding fragment thereof of claim 1, which is an antibody which is a monoclonal antibody, a labeled antibody, a bivalent antibody, a polyclonal antibody, a bispecific antibody, a chimeric antibody, a recombinant antibody, an anti-idiotypic antibody, a humanized antibody or a bispecific antibody.

8. The antibody or antigen-binding fragment thereof of claim 1, which is an antigen-binding fragment which is a camelized single domain antibody, a diabody, an scfv, an scfv dimer, a dsfv, a (dsfv)₂, a dsFv-dsfv′

, a bispecific ds diabody, an Fv, an Fab, an Fab′

, an F(ab′

)₂, or a domain antibody.

9. The polypeptide of claim 2 which is linked to an immunoglobulin constant region polypeptide.

10. The polypeptide of claim 9 wherein the constant region polypeptide is a γ

1 heavy chain, γ

2 heavy chain, γ

3 heavy chain or γ

4 heavy chain.

12. An isolated polypeptide of claim 1 comprising a member selected from the group consisting of:

(i) HCDR1 comprising the amino acid sequence G F N I K D T Y M H (SEQ ID NO;

     11), HCDR2 comprising the amino acid sequence R I D P A N G H T E Y D P K F Q D (SEQ ID NO;

     12) and HCDR3 comprising the amino acid sequence S Y F G S I F A Y (SEQ ID NO;

     13);

(ii) HCDR1 comprising the amino acid sequence G F N I K D T Y I H (SEQ ID NO;

     19), HCDR2 comprising the amino acid sequence R I D P A N G H T E Y D P K F Q G (SEQ ID NO;

     20) and HCDR3 comprising the amino acid sequence S Y Y G S I F A Y (SEQ ID NO;

     21);

(iii) HCDR1 comprising the amino acid sequence G F N I K D Y Y I H (SEQ ID NO;

     27), HCDR2 comprising the amino acid sequence W I D P E N G D T E Y A P K F Q G (SEQ ID NO;

     28) and HCDR3 comprising the amino acid sequence Y Y R Y D D G T W F P Y (SEQ ID NO;

     29);

(iv) HCDR1 comprising the amino acid sequence G F N I K D T Y I H (SEQ ID NO;

     35), HCDR2 comprising the amino acid sequence W I D P A N G Y T K Y A P N F Q G (SEQ ID NO;

     36) and HCDR3 comprising the amino acid sequence G Y Y R Y Y S L D Y (SEQ ID NO;

     37);

(v) HCDR1 comprising the amino acid sequence G F T F S N H D M A (SEQ ID NO;

     43), HCDR2 comprising the amino acid sequence S I T P S G G T T Y Y R D S V E G (SEQ ID NO;

     44) and HCDR3 comprising the amino acid sequence Q N Y Y D G S Y Y Y G L Y Y F D Y (SEQ ID NO;

     45);

(vi) HCDR1 comprising the amino acid sequence G Y T F T D Y Y M N (SEQ ID NO;

     51), HCDR2 comprising the amino acid sequence D I N P N N G G A I Y N Q K F K G (SEQ ID NO;

     52) and HCDR3 comprising the amino acid sequence G I I T E I A E D F (SEQ ID NO;

     53);

(vii) LCDR1 comprising the amino acid sequence S A S S S V S Y L Y (SEQ ID NO;

     15), LCDR2 comprising the amino acid sequence R S S H R A S (SEQ ID NO;

     16) and LCDR3 comprising the amino acid sequence H Q Y Q S Y P P T (SEQ ID NO;

     17);

(viii) LCDR1 comprising the amino acid sequence S A S S S V S Y L F (SEQ ID NO;

     23), LCDR2 comprising the amino acid sequence R T S Y L A S (SEQ ID NO;

     24) and LCDR3 comprising the amino acid sequence H Q Y H T Y P P T (SEQ ID NO;

     25);

(ix) LCDR1 comprising the amino acid sequence R A S G N I H S Y L A (SEQ ID NO;

     31), LCDR2 comprising the amino acid sequence N A K T L P D (SEQ ID NO;

     32) and LCDR3 comprising the amino acid sequence Q H F W N T P W T (SEQ ID NO;

     33);

(x) LCDR1 comprising the amino acid sequence R A S Q D I S N Y L N (SEQ ID NO;

     39), LCDR2 comprising the amino acid sequence Y S S R L H S (SEQ ID NO;

     40) and LCDR3 comprising the amino acid sequence Q Q G K T L P L T (SEQ ID NO;

     41);

(xi) LCDR1 comprising the amino acid sequence R S S Q S L V Y S D G N T Y L H (SEQ ID NO;

     47), LCDR2 comprising the amino acid sequence R V S N R F S (SEQ ID NO;

     48) and LCDR3 comprising the amino acid sequence L Q S T H F P P T (SEQ ID NO;

     49); and

(xii) LCDR1 comprising the amino acid sequence K A S Q N V G T N V V (SEQ ID NO;

     55), LCDR2 comprising the amino acid sequence S A S Y R Y S (SEQ ID NO;

     56) and LCDR3 comprising the amino acid sequence Q Q Y K T Y P Y T (SEQ ID NO;

     57).

13. The polypeptide of claim 12 comprising HCDR1 comprising the amino acid sequence G Y T F T D Y Y M N (SEQ ID NO:

51), HCDR2 comprising the amino acid sequence D I N P N N G G A I Y N Q K F K G (SEQ ID NO;

     52) and HCDR3 comprising the amino acid sequence G I I T E I A E D F (SEQ ID NO;

     53).

17. An isolated antibody or antigen-binding fragment thereof comprising the polypeptide of claim 13.

18. The antibody or antigen-binding fragment thereof of claim 17, which is a humanized antibody.

19. The antibody or antigen-binding fragment thereof of claim 17, which is an antibody which is a monoclonal antibody, a labeled antibody, a bivalent antibody, a polyclonal antibody, a bispecific antibody, a chimeric antibody, a recombinant antibody, an anti-idiotypic antibody, a humanized antibody or a bispecific antibody.

20. The antibody or antigen-binding fragment thereof of claim 17, which is an antigen-binding fragment which is a camelized single domain antibody, a diabody, an scfv, an scfv dimer, a dsfv, a (dsfv)₂, a dsFv-dsfv′

, a bispecific ds diabody, an Fv, an Fab, an Fab′

, an F(ab′

)₂, or a domain antibody.

21. The polypeptide of claim 13 which is linked to an immunoglobulin constant region polypeptide.

22. The polypeptide of claim 21 wherein the constant region polypeptide is a γ

1 heavy chain, γ

2 heavy chain, γ

3 heavy chain or γ

4 heavy chain.

24. The polypeptide of claim 1 comprising a member selected from the group consisting of:

(a) the amino acid sequence;

25. The polypeptide of claim 24 comprising the amino acid sequence:

E V Q L Q Q S G

29. An isolated antibody or antigen-binding fragment thereof comprising the polypeptide of claim 25.

30. The antibody or antigen-binding fragment thereof of claim 29, which is a humanized antibody.

31. The antibody or antigen-binding fragment thereof of claim 29, which is an antibody which is a monoclonal antibody, a labeled antibody, a bivalent antibody, a polyclonal antibody, a bispecific antibody, a chimeric antibody, a recombinant antibody, an anti-idiotypic antibody, a humanized antibody or a bispecific antibody.

32. The antibody or antigen-binding fragment thereof of claim 29, which is an antigen-binding fragment which is a camelized single domain antibody, a diabody, an scfv, an scfv dimer, a dsfv, a (dsfv)₂, a dsFv-dsfv′

, a bispecific ds diabody, an Fv, an Fab, an Fab′

, an F(ab′

)₂, or a domain antibody.

33. The polypeptide of claim 25 which is linked to an immunoglobulin constant region polypeptide.

34. The polypeptide of claim 33 wherein the constant region polypeptide is a γ

2 heavy chain.

35. An isolated polynucleotide encoding the polypeptide of claim 1.

36. An isolated vector comprising the polynucleotide of claim 35.

37. An isolated host cell comprising the polynucleotide of claim 35.

40. A method for reducing:

total cholesterol level;

low density lipoprotein cholesterol level;

apolipoprotein B level;

total cholesterol/high density lipoprotein ratio;

or

low density lipoprotein/high density lipoprotein ratio;

in a subject, comprising administering, to said subject, a therapeutically effective amount an antibody or antigen-binding fragment thereof of claim 6, that binds specifically to PCSK9;

which antibody or fragment inhibits binding between PCSK9 and LDL receptor;

optionally in association with a further chemotherapeutic agent.

42. A method for treating or preventing hypercholesterolemia, hyperlipidemia, hypertriglyceridaemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, vascular inflammation or xanthoma, in a subject, comprising administering, to said subject, a therapeutically effective amount of an antibody or antigen-binding fragment thereof of claim 6, that binds specifically to PCSK9, which antibody or fragment inhibits binding between PCSK9 and LDL receptor;

optionally in association with a further chemotherapeutic agent.

44. A method for reducing:

total cholesterol level;

low density lipoprotein cholesterol level;

apolipoprotein B level;

total cholesterol/high density lipoprotein ratio;

or

low density lipoprotein/high density lipoprotein ratio;

or

for treating;

hypercholesterolemia;

hyperlipidemia;

hypertriglyceridaemia;

sitosterolemia;

atherosclerosis;

arteriosclerosis;

coronary heart disease;

vascular inflammation;

or

xanthoma,

in a subject, comprising administering, to said subject, a therapeutically effective amount of the antibody or antigen-binding fragment thereof of claim 6.

45. The method of claim 44 wherein the antibody or antigen-binding fragment thereof is administered in association with a further chemotherapeutic agent.

46. The method of claim 45 wherein the further chemotherapeutic agent is a cardiovascular agent, an adrenergic blocker, an antihypertensive agent, an angiotensin system inhibitor, an angiotensin-converting enzyme inhibitor, a coronary vasodilator, a diuretic, an adrenergic stimulant or an HMG-CoA reductase inhibitor.

47. The method of claim 45 wherein the further chemotherapeutic agent is ezetimibe, lovastatin, atorvastatin, pravastatin, rosuvastatin, fluvastatin, rivastatin, simvastatin, an azetidinone, bunolol hydrochloride, acebutolol, alprenolol hydrochloride, atenolol, carteolol hydrochloride, celiprolol hydrochloride;

cetamolol hydrochloride, labetalol hydrochloride, esmolol hydrochloride, levobetaxolol hydrochloride, levobunolol hydrochloride, nadolol, practolol, propranolol hydrochloride, sotalol hydrochloride, timolol, timolol maleate, bisoprolol;

bisoprolol fumarate, nebivalol, cicloprolol hydrochloride, dexpropranolol hydrochloride, diacetolol hydrochloride, dilevalol hydrochloride, exaprolol hydrochloride, flestolol sulfate, metalol hydrochloride, metoprolol 2-Propanol, metoprolol tartrate, pamatolol sulfate, penbutolol sulfate, practolol, tiprenolol hydrochloride, tolamolol tenspiride hydrochloride, proroxan, alfuzosin hydrochloride, labetalol hydrochloride, bretylium tosylate, dihydroergtamine mesylate, carvedilol, labetalol, bretylium tosylate, phentolamine mesylate, solypertine tartrate, zolertine hydrochloride, candesartan cilexetil, telmisartan, candesartan;

losartan potassium, benazepril hydrochloride, captopril, fosinopril, moexipril hydrochloride, perindopril erbumine, quinapril, ramipril, enalapril maleate, lisinopril, delapril, spirapril, delapril hydrochloride, libenzapril, pentopril, perindopril 1H-Indole-2-carboxylic acid, quinaprilat, spirapril hydrochloride, spiraprilat, teprotide, zofenopril, amlodipine besylate, clentiazem maleate, isradipine, nimodipine, felodipine, nilvadipine;

diltiazem hydrochloride, verapamil hydrochloride, teludipine hydrochloride, belfosdil, fostedil, ranolazine, butoprozine hydrochloride, tosifen, molsidomine, ranolazine hydrochloride, tosifen, diltiazem hydrochloride, isosorbide dinitrate, sosorbide mononitrate, nitroglycerin, verapamil hydrochloride, chromonar, clonitate, droprenilamine, lidoflazine, prenylamine, propatyl nitrate, mioflazine hydrochloride, mixidine, molsidomine, isosorbide mononitrate, erythrityl tetranitrate, clonitrate, dipyridamole;

nicorandil, pyridinecarboxamide, nifedipine;

perhexyline maleate;

oxprenolol, pentrinitrol, verapamil, althiazide, benzthiazide, buthiazide, chlorothiazide, spironolactone, triamterene, guanfacine hydrochloride, methyldopa-hydrochlorothiazide combined with Hydrochlorothiazide, methyldopa-chlorothiazide, clonidine hydrochloride, chlorthalidone, clonidine hydrochloride, clonidine, althiazide, benzthiazide, captopril, carvedilol, chlorothiazide, clonidine hydrochloride, cyclothiazide, delapril hydrochloride, dilevalol hydrochloride, delapril hydrochloride, doxazosin mesylate, fosinopril sodium, moexipril hydrochloride, monatepil maleate, metoprolol succinate, guanfacine hydrochloride, methyldopa, quinaprilat, quinapril hydrochloride, primidolol, prazosin hydrochloride, pelanserin hydrochloride, phenoxybenzamine hydrochloride, candesartan cilexetil, telmisartan, candesartan, amlodipine maleate, terazosin hydrochloride, bevantolol hydrochloride or ramipril.

48. The method of claim 47 wherein the further chemotherapeutic agent is ezetimibe and, optionally, simvastatin.

49. The method of claim 44 wherein the antibody or antigen-binding fragment thereof is a humanized antibody.

56. A method for producing a polypeptide of claim 1 comprising introducing a polynucleotide into a host cell;

which polynucleotide encodes and directs expression of said polypeptide; and

growing said host cell under conditions whereby said polypeptide is expressed; and

, optionally, isolating the polypeptide.

57. The method of claim 56 wherein the host cell is a Chinese hamster ovary cell.

58. The method of claim 56 wherein the host cell is a bacterial cell.

59. The method of claim 56 wherein the host cell is a fungal cell.

60. The method of claim 56 wherein the host cell is a Pichia cell.

61. The method of claim 56 wherein the host cell is a Pichia pastoris cell.

3-5. -5. (canceled)

11. (canceled)

14-16. -16. (canceled)

23. (canceled)

26-28. -28. (canceled)

38. An isolated polypeptide comprising an amino acid sequence comprising about 90% or more amino acid sequence similarity to a fragment of the human LDL receptor which fragment consists of amino acids beginning at about amino acid position 314 and ending at about amino acid position 355 of said receptor;

wherein said polypeptide optionally comprises one or more properties selected from the group consisting of;

(i) binds to PCSK9;

(ii) competes with LDL receptor or an anti-PCSK9 antibody or antigen-binding fragment thereof for binding to PCSK9;

(iii) reduces total cholesterol level when administered to an animal;

(iv) reduces low density lipoprotein cholesterol level when administered to an animal;

(v) reduces apolipoprotein B level when administered to an animal;

(vi) reduces total cholesterol/high density lipoprotein ratio when administered to an animal; and

(vii) reduces low density lipoprotein/high density lipoprotein ratio when administered to an animal;

or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier.

39. The polypeptide of claim 38 which consists of the amino acid sequence of SEQ ID NO:

3;

or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier.

41. (canceled)

43. (canceled)

50-55. -55. (canceled)